A vascular endothelial growth factor (VEGF) is a signaling protein which plays an important role in promoting both vasculogenesis and angiogenesis. The VEGF functions as a part of the system which restores and supplies oxygen to tissues, when blood circulation is inadequate. The well-known function of the VEGF is to create new blood vessels during embryonic development, at the injured site, in muscle following exercise, and in the formation of vessels to bypass blocked vessels.
The increased amount of VEGFs, however, may cause abnormal angiogenesis. In particular, the angiogenesis is related to tumor development such that it is involved in primary or metastatic tumor generation. Further, cancer metastasis occurs when a primary cancer cell is introduced into blood vessel by the angiogensis. It is reported that the angiogenesis causes, for example, asthma, difficulty in breathing, endometriosis; acute and chronic inflammation, such as, atherosclerosis and tissue edema; infectious disease, such as, hepatitis and Kaposis sarcoma; autoimmune disease, such as, diabetes, psoriasis, rheumarthritis and thyroiditis; and other diseases comprising diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), angiofibroma, and etc. (Carmeliet, P. y Jain, R K. 2000. Nature 407: 249-257; Kuwano M, et al. 2001. Intern Med 40: 565-572).
The VEGF, which plays an important role in angiogenesis, influences mainly on the vessel endothelium cells. In accordance with in vitro test, the VEGF stimulates division and wandering of the endothelial cells, and increases capillary vessel permeability as well.
In mammals, VEGF is classified into 5 types, such as, VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF (placenta growth factor). The VEGF-A stimulates angiogenesis, endothelial cell wandering, division, blood vessel lumen formation, chemotaxis of macrophage and granulocyte, and blood vessel expansion. The VEGF-B promotes embryonic angiogenesis, in particular, the development of myocardial tissues. The VEGF-C accelerates lymphangiogenesis, and the VEGF-D is necessary for the growth of lymphatic vessels which enclose the lung bronchioles. Further, the PIGF is important for angiogenesis in the vasculogenesis, ischemia, inflammation, injury recovery, and cancer.
The VEGF receptor (VEGFR), a receptor whose ligand is VEGF, consists of 3 subtypes (VEGFR1 (flt-1), VEGFR2 (Kdr/flk-1) and VEGFR3 (flt-4)), and exists in the form of membrane-bound VEGFR (mbVEGFR) or soluble VEGFR (sVEGFR) through the alternative splicing of genes.
The VEGFR is expressed in neurons, Kaposi's sarcoma, and hematopoietic stem cells, while it is usually found in endothelial cells. Once the VEGF binds to VEGFR, signals are transferred by dimerization of the receptor and phosphorylation of the receptor tyrosine residues.
The VEGF binds to its receptors, VEGFR1 and VEGFR2, with high affinity and transfers signals mainly through the VEGFR2, and induces the mechanism of angiogenesis comprising growth and migration of endothelial cells.
Therefore, VEGF and VEGFR2 have been studied as targets for inhibiting or suppressing angiogenesis induced by the VEGF (Ellis and Hicklin, Nature Rev. Cancer, 8:579, 2008; Youssoufian et al., Clin. Cancer Res., 13:5544s, 2007).
The bispecific antibody or multi-specific antibody reported in the previous studies includes i) Conbercept consisting of VEGFR1 domain2, VEGFR2 domain 3, domain4 and IgG1 Fc, ii) VEGF-Trap (aflibercept) consisting of VEGFR1 domain2, VEGFR2 domain3 and IgG1 Fc, and iii) VEGF-Grab consisting of VEGFR1 domain2, domain3 and IgG1 Fc.
We, inventors, have studied eagerly to develop a multi-specific antibody for treating cancers and ocular diseases by suppressing or inhibiting angiogenesis, and have surprisingly found that the fusion protein construct of immunoglobulin-like domain of the present invention binds to VEGF-A and PIGF with both high sensitivity and specificity, and has significantly excellent effects of inhibiting proliferation, growth and/or angiogenesis of various tumors.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.